Rapid screening of mtDNA coding region SNPs for haplogroup identification of forsensic samples

In the last few years, the analysis of mitochondrial DNA (mtDNA) has greatly gained in importance. The high copy number, the elevated stability against degradation and the strict maternal inheritance are the main characteristics of the mitochondrial genome, which make it particularly suitable for palaeogenetic inferences and the reconstruction of human evolution. In forensic DNA fingerprinting, mtDNA profiling is established as a technological resource for cases where conventional nuclear DNA markers fail to give satisfactory results (e.g. analyses of hair shafts, remains of bones and teeth). The increasing availability of large-scale mtDNA databases has made it possible to ask detailed questions about the structure of human genetic diversity, and what that structure can teach us about human demographic history. Global, multi-locus analyses have suggested that human genetic diversity may fall into clusters that correspond approximately to continental origin. Such clusters of evolutionary related lineages are called haplogroups. In this grant proposal, two new multiplex systems are introduced, which are based on screening SNPs from the mitochondrial DNA coding region via the concept of simultaneous PCR amplification of several gene segments and simultaneous minisequencing reactions for the visualization of the allelic status of haplogroup diagnostic SNPs. The multiplex systems are aimed to serve the forensic investigator in several important ways: as screening tools, the assays provide quick information for the elimination of exclusionary samples; for missing person identifications especially after mass disasters, kinship analyses can be performed on highly degraded samples and with even distant maternal relatives as reference samples; as a dissection tool, one of the two assays supplies further discrimination of samples belonging to the second most common European haplogroup cluster that cannot be discriminated with control region sequencing. The proposed multiplex systems pose a new and innovative tool for forensic casework and human evolution studies, which become very effective in investigations involving a high number of samples and samples belonging to the second most common European haplogroup cluster.